Process for producing d-alpha-alanine and/or l-alpha-alanineamide by arthrobacter sp

ABSTRACT

A novel D-amidase is described. The enzyme specifically hydrolyzes D-α-alanineamide into D-α-alanine. It is produced by culturing a microorganism belonging to the genus Arthrobacter, and is useful as an enzyme for efficiently producing D-α-alanine having a high optical purity and/or L-α-alanineamide from DL-α-alanineamide or D-α-alanineamide at low cost.

This application is a continuation of application Ser. No. 07/327,000filed Mar. 22, 1989, now abandoned.

BACKGROUND OF THE INVENTION

The present invention relates to a novel enzyme capable of specificallyhydrolyzing D-α-alanineamide to produce D-α-alanine (hereinafterreferred to as D-amidase) and a process for producing D-α-alanine and/orL-α-alanineamide using the enzyme.

D-α-alanine is an important compound as a sweetening, or a syntheticintermediate or starting compound for synthesizing variousphysiologically active substances. L-α-Alanineamide is a startingcompound for producing L-α-alanine, which is an important amino acid asfood and medicines.

The following processes for producing D-α-alanine have been known sofar.

(1) A microbiological process for producing D-α-alanine through directfermentation [Japanese Published Unexamined Patent Application Nos.22881/76 and 76482/77].

(2) A process for producing D-α-alanine by separating D-α-alanine fromDL-α-alanine through the action of microorganism having an ability todecompose only L-α-alanine [Ohshima and Tanaka: Amino Acid Nucleic Acid,15, 89-93 (1966)].

(3) A process for producing D-α-alanine through the action of acylaseproduced by a microorganism, upon the N-acyl compound of DL-α-alanineand through the successive optical resolution of DL-α-alanine (JapanesePublished Examined Patent Application No. 22380/66).

(4) A process for producing D-α-alanine through the action ofmicroorganisms having a hydantoinase activity upon 5-methylhydantoin toform D-(N-carbamoyl)alanine, and by treating D-(N-carbamoyl)alaninechemically or microbiologically [Yamada, et al: Hakko To Kogyo, 38, 937(1980), Japanese Published Unexamined Patent Application Nos. 91189/78,89088/79,88697/80, 104890/80, 114291/80, etc.].

(5) A process for producing D-α-alanine by hydrolyzing D-α-alanineamidethrough the hydrolytic activity possessed by microorganisms belonging tothe genus Bacillus, Bacterium, Micrococcus, Brevibacterium,Achromobacter, Alcaligenes, Kurthia, Pseudomonas, Rhodococcus orSerratia [Japanese Published National Publication No. 500319/81,Japanese Published Unexamined Patent Application Nos. 184392/85,96989/86 and 274690/86].

(6) A process for producing D-α-amino acid by hydrolyzing DL-α-aminoacid amide through the hydrolytic activity specific to D-α-amino acidamide and possessed by microorganisms belonging to the genus Rhodococcus[Japanese Published Unexamined Patent Application No. 87998/88].

(7) A process for producing D-α-alanine through the action ofD-amino-acid transaminase upon pyruvic acid [Japanese PublishedUnexamined Patent Application No. 205790/87].

(8) A process for producing D-α-alanine by the chemical opticalresolution through the preferential crystallization of DL-α-alaninep-chlorobenzenesulfonate [Japanese Published Examined Patent ApplicationNo. 14369/72 and Japanese Published Unexamined Patent Application No.57914/73].

Among the aforementioned processes for producing D-α-alanine, theprocesses (1), (2), (6) and (8) have no or lower D-α-alanineproductivity, and the processes (3), (4) and (7) have complicatedoperations because of the reaction consisting of several stages. Theprocesses (5) and (7) require an expensive optically active substrateand thus raise the cost of production.

The enzymes capable of hydrolyzing D-α-alanineamide are disclosed inAbstracts of 1988th Meeting of the Agricultural Chemical Society ofJapan, page 352.

Any process for producing L-α-alanineamide of high optical purity atindustrially low cost is unknown yet.

Presently, an enzyme capable of producing D-α-alanine andL-α-alanineamide of high optical purity directly from inexpensiveDL-α-alanineamide and a process for producing D-α-alanine andL-α-alanineamide using the enzyme are desired.

Extensive studies have been made on the development of an industriallyfavorable process for producing D-α-alanine from DL-α-alanineamide. As aresult, the present inventors have found that microorganisms belongingto the genus Arthrobacter produce an enzyme capable of specificallyhydrolyzing D-α-alanineamide thereby to produce D-α-alanine fromDL-α-alanineamide or D-α-alanineamide. Examination of itsphysicochemical properties after isolation and purification of theenzyme reveals that the enzyme is novel.

SUMMARY OF THE INVENTION

The present invention provides a novel D-amidase as well as a processfor producing D-α-alanine and/or L-α-alanineamide, which comprisescarrying out an enzymatic hydrolysis in an aqueous medium containingDL-α-alanineamide or D-α-alanineamide in the presence of a culture,cells, a treated product thereof or D-amidase isolated therefrom, of amicroorganism belonging to the genus Arthrobacter and capable ofproducing D-amidase, and recovering D-α-alanine and/or L-α-alanineamidefrom the resulting reaction mixture.

The physicochemical properties of the novel D-amidase are given below.

1) Activity and substrate specificity: It specifically hydrolyzesD-α-alanineamide into D-α-alanine; the activity of hydrolyzingL-α-alanineamide is 0 to 1.5% of the activity of hydrolyzingD-α-alanineamide.

2) Optimum pH: pH 7 to 8 at 30° C.

3) Optimum temperature: 40° to 45° C. at pH of 7.5

4) Heat stability: It is inactivated when allowed to stand at atemperature above 60° C. for 10 minutes.

5) pH stability: It is stable within a range of pH 6.5 to 10 at 30° C.

6) Molecular weight: 50,000±5,000 (SDS-polyacrylamide electrophoresis)

7) Activation: No coenzyme is required for activation.

8) Isoelectric point: pH 5.2±0.3

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the relative activity with change in pH, when defining theenzyme activity at pH 7.5 as 100%.

FIG. 2 shows the relative activity with change in temperature, whendefining the enzyme activity at 37° C. as 100%.

FIG. 3 shows the pH stability of the enzyme.

FIG. 4 shows the heat stability of the enzyme.

DESCRIPTION OF THE INVENTION

Any microorganisms can be used in producing of the enzyme of the presentinvention, so far as they belong to the genus Arthrobacter and have anability to produce the enzyme having the properties described above. Forexample, Arthrobacter sp. H-4904 can be mentioned.

Arthrobacter sp. H-4904 is a microorganism newly isolated from thenatural source.

Bacteriological properties of Arthrobacter sp. H-4904 are describedbelow:

(a) Morphology

1) Form and size of cells: Spherical form (0.8-1.0 μm in diameter) androd form (0.8 μm in diameter and 1.2-1.5 μm long).

Motility: Motile with flagella occurring in polar positions.

3) Spore: none

4) Gram staining: positive

5) Acid resistance: scarcely observed

(b) Growth states on various media

1) Bouillon-agar plate culture forms circular, convex, smooth colonies,showing an opaque, whitish yellow color without formation of diffusiblepigments.

2) Bouillon-agar slant culture gives well growth, showing an opaque,whitish yellow color.

3) Bouillon liquid culture gives turbid growth without formation ofsurface membrane.

4) Bouillon-gelatin stab culture gives no liquefaction.

5) Litmus milk: no reduction of litmus and no coagulation.

(c) Physiological properties

1) Nitrate reduction: positive

2) Denitrification reaction: positive

3) MR test: negative

4) VP test: negative

5) Indole formation: negative

6) Hydrogen sulfide formation: negative

7) Starch hydrolysis: negative

8) Citric acid utilization (Simons' medium): positive

9) Utilization of inorganic nitrogen source

Nitrate: negative

Ammonium salt: weakly positive

10) Pigment formation: negative

11) Urease: negative

12) Oxidase: negative

13) Catalase: positive

14) Growth range

1) pH: 5.0-9.0 (optimum pH: 6.0-8.0)

2) Temperature: 15°-37° C. (optimum temperature: 30° C.)

15) Oxygen relationship: aerobe or facultative anaerobe

16) OF test: negative

17) Acid and gas formation from saccharides:

    ______________________________________                                                    Acid Gas (peptone-water)                                          ______________________________________                                        L-arabinose   --     --                                                       D-xylose      --     --                                                       D-glucose     --     --                                                       D-mannose     --     --                                                       D-fructose    --     --                                                       D-galactose   --     --                                                       Maltose       --     --                                                       Sucrose       --     --                                                       Lactose       --     --                                                       Trehalose     --     --                                                       D-sorbitol    --     --                                                       D-mannitol    --     --                                                       Inositol      --     --                                                       Glycerine     --     --                                                       Starch        --     --                                                       ______________________________________                                    

18) Sodium chloride resistance: grow at 15% NaCl.

(d) Chemical Composition

1) Peptidoglycan-constituting amino acids: lysine, alanine and glutamicacid

2) mol % G+C (Tm): 63.17

The microorganism with the aforementioned bacteriological properties wasidentified as bacteria belonging to the genus Arthrobacter, referring toBergey's Manual of Systematic Bacteriology, Vol.2 (1986) on the basis ofsuch properties as gram-positive spherical or rod form; motile withflagella occurring in polar positions, aerobe or facultative anaerobe;non-formation of spores; lysine, alanine and glutamic acid aspeptidoglycan-constituting amino acids; mol % G+C of DNA being 63.17.The strain was named Arthrobacter sp. H-4904 and has been deposited withthe Fermentation Research Institute (FRI), the Agency of IndustrialScience and Technology under the Budapest Treaty since Jan. 14, 1988with the accession number of FERM BP-1649.

Any of natural media and synthetic media can be used as the medium forculturing the microorganism, so far as they appropriately contain acarbon source, a nitrogen source, inorganic salts, etc. that can beassimilated by the microorganism and that the microorganism having anability to produce the enzyme of the present invention can beefficiently cultured therein.

As the carbon source, carbohydrates such as glucose, sucrose, molasses,starch hydrolyzate, etc., organic acids such as acetic acid, propionicacid, etc., and alcohols such as ethanol, propanol, etc. can be used.

As the nitrogen source, ammonia, various ammonium salts of inorganic andorganic acids such as ammonium chloride, ammonium sulfate, ammoniumacetate, ammonium phosphate, etc., amines and other nitrogen-containingcompounds, peptone, meat extract, yeast extract, corn steep liquor,casein hydrolyzate, soybean cake hydrolyzate, various fermentation cellsand their digested products, etc. can be used.

As the inorganic materials, potassium dihydrogen phosphate, dipotassiumhydrogen phosphate, magnesium phosphate, magnesium sulfate, sodiumchloride, ferrous sulfate, manganese sulfate, copper sulfate, calciumcarbonate, etc. can be used.

Culturing is carried out under aerobic conditions with shaking,submerged aeration stirring, etc. preferably at a temperature of 15° to37° C., usually for 16-72 hours, while keeping pH at 5.0-9.0 by addingan inorganic or organic acid, an alkaline solution, urea, calciumcarbonate, ammonia, etc.

It is necessary to add 0.1-20 g/l D-α-alanineamide, L-α-alanineamide orDL-α-alanineamide to the medium in the culturing, for the purpose ofinducing and accumulating D-amidase. By use of an appropriate mutant,D-amidase can be accumulated without adding D-α-alanineamide,L-α-alanineamide or DL-α-alanineamide (which is hereinafter referred toas "inducing substance") to the medium.

The mutant can be obtained from Arthrobacter sp. H-4904 as the parentstrain according to the ordinary mutagenesis, for example, throughultraviolet irradiation, radiation irradiation such as X-rayirradiation, treatment with mutation-inducing agent, etc.

After the mutation treatment, a desired mutant can be obtained byrecovering colonies growing in an ordinary nutrient medium, for example,a bouillon-yeast extract medium and selecting a strain that accumulatesD-amidase in a medium containing no inducing substance.

An example of the thus obtained mutant is Arthrobacter sp. H-7095, whichhas been deposited with the FRI under the Budapest Treaty since Mar. 2,1988 with the accession number of FERM BP-1773.

In order to recover and purify the enzyme from the culture, conventionalpurification methods for an enzyme may be used. For example, the cultureis centrifuged and the cells are collected. The collected cells aredisrupted by means of mechanical disruption using a DYNO-MILL, FrenchPress, Manton-Gaulin homogenizer, ultrasonication, etc., and theresulting suspension is centrifuged to remove cell debris. The enzyme inthe supernatant is isolated and purified by salting-out method usingammonium sulfate, ion exchange chromatography using DEAE-Sepharose,CM-Sepharose, etc., whereby an SDS-polyacrylamide gelelectrophoretically pure enzyme can be obtained.

The enzyme activity is determined as follows.

After 1.0 ml of 50 mM phosphate buffer (pH 7.5) containing 250 mMD-α-alanineamide is heated at 30° C. for 5 minutes, 0.1 ml of an enzymesolution is added thereto to carry out incubation at 30° C. for 30minutes. The reaction is terminated by adding 0.1 ml of 6N hydrochloricacid thereto. The amount of the D-α-alanine formed during the reactionis determined by high performance liquid chromatography (HPLC) under thefollowing conditions.

Column CHIRALPAK WE(-), manufactured by Daicel Ltd.

Eluate: 0.25 mM CuSO₄ aqueous solution

Flow rate: 1 ml/min

Column temperature: 45° C.

Method for detection: o-Phthalaldehyde is added to react at 50° C. andfluorescence is detected (excitation wavelength: 344 nm; fluorescentwavelength: 444 nm)

The enzyme activity is defined as 1 unit (U) in terms of the activityfor hydrolyzing D-α-alanineamide into 1 μmol of D-α-alanine for a minuteunder the assay conditions defined above.

The activity for hydrolyzing L-α-alanineamide into L-α-alanine(hereafter referred to as L-amidase activity) can be determined underthe conditions described above except that L-α-alanineamide is used inplace of D-α-alanineamide.

Though D-α-alanine and/or L-α-alanineamide can be obtained by acting theenzyme of the present invention on DL-α-alanineamide orD-α-alanineamide, it is preferred that an enzymatic hydrolysis iscarried out in an aqueous medium containing DL-α-alanineamide orD-α-alanineamide in the presence of a culture, cells or a treatedproduct thereof, of the microorganism belonging to the genusArthrobacter and capable of producing the enzyme of the presentinvention, and that D-α-alanine and/or L-α-alanineamide can be recoveredfrom the resulting reaction mixture.

The enzymatic hydrolysis can be carried out either during the culturingof the microorganism or by allowing the culture, cells, a treatedproduct thereof, or the purified enzyme to react DL-α-alanineamide orD-α-alanineamide in an aqueous medium after the completion of culturing.

The treated product of cells of the microorganism having the D-amidaseactivity includes, for example, dried cells, freeze-dried cells,surfactant-treated cells, enzyme-treated cells, ultrasonically disruptedcells, mechanically disrupted cells, solvent-treated cells, andproteinous fractions of cells, and further includes immobilized productsof cells and the aforesaid treated products thereof. Purified D-amidasemay be used and may also be used as the immobilized products.

The aqueous medium includes, for example, water, buffer solutions ofphosphate, carbonate, acetate, borate, citrate, Tris, etc., alcoholssuch as methanol, ethanol, propanol, etc., esters such as ethyl acetate,etc., ketones such as acetone, etc., and amides such as acetamide, etc.

When purified enzyme is used in the reaction, it is preferred that 5 to50% glycerol is added to the aqueous medium in order to improve thestability of the enzyme.

The reaction is carried out usually at a temperature of 15° to 50° C.and pH of 6.0 to 9.5 for 1 to 72 hours. The potency of enzyme in thereaction mixture depends on the amount of DL-α-alanineamide orD-α-alanineamide used and the reaction time, and usually is 1 to 300kU/l. Specifically, when cells are used in the reaction, theconcentration of cells is usually 1 to 50 g/l as wet cells. TheDL-α-alanineamide or D-α-alanineamide for use in the reaction can be inany one of free form, hydrochloride form and sulfate form. In case ofDL-α-alanineamide, 1-500 g/l, preferably 1-400 g/l can be used, and incase of D-α-alanineamide, 1-300 g/l, preferably 1-200 g/l can be used.

The alanine racemase usually contained in microbial cells is an enzymethat catalyzes racemation of optically active alanine and lowers theoptical purity of D-α-alanine formed in the process of the presentinvention. The microorganism to be used in the present invention has asmaller alanine racemase content, and thus D-α-alanine of satisfactorilyoptical purity can be obtained by use of the microorganisms of thepresent invention as such. However, D-α-alanine of much higher opticalpurity can be obtained by appropriately using well known procedures forsuppressing the alanine racemase activity, for example, by obtaining amutant with no or less alanine racemase activity through an ordinarymutagenesis [J. Wild, et al.: Mol. Gen. Genet. 198, 315-322 (1985)]; byinactivating the activity of alanine racemase in the microorganismthrough a heat treatment, etc. [Takamatsu, Tosa and Chihata J. Jap.Chem. Soc. 9 1369 (1983)], and by adding an alanine racemase inhibitorto the aqueous medium during the reaction [Kagaku To Seibutsu 20 770-772(1986); Seikagaku Jikken Koza 11 275-296].

When DL-α-alanineamide is used in the enzymatic hydrolysis, D-α-alanineis formed and accumulated in the aqueous medium and concurrently,L-α-alanineamide remains in the aqueous medium after the hydrolysis.Thus, L-α-alanineamide can be obtained by recovering it from the aqueousmedium.

D-α-alanine and L-α-alanineamide can be recovered from the culture orthe aqueous medium according to an ordinary separation procedure, forexample, through column chromatography using ion exchange resin, etc.,or through crystallization.

The present invention is described below, referring to Examples.

EXAMPLE 1

At first, 150 ml of BYG medium [a medium containing 2% of bouillonpowder (product of Kyokuto Co.), 0.5% of yeast extract (product ofDifco), 0.5% of polypeptone and 0.2% of glucose, adjusted to pH 7.2 with6N NaOH] was poured into each of 2 l-flasks provided with baffles, andsterilized at 120° C. for 20 minutes. Then, one loopful of Arthrobactersp. H-4904 growing on a bouillon slant was inoculated into each of themedia and cultured at 30° C. for 20 hours with shaking. The resultingculture was used as a seed culture.

Separately, a medium containing 3% of glucose, 2% of corn steep liquor,0.5% of peptone, 1% of NaCl, 2% of (NH₄)₂ SO₄, 0.3% of MgSO₄.7H₂ O,0.001% of FeSO₄.7H₂ O, 0.0001% of MnSO₄.7H₂ O and 0.6% ofDL-α-alanineamide and having a pH of 7.2 was prepared, and 18 l of thethus prepared medium was poured into a 30 l-jar fermenter and sterilizedat 120° C. for 20 minutes. Then, 2 l of the seed culture was asepticallyintroduced into the medium and cultured with agitation and aeration (450rpm, 10 l/min) at 30° C. for 30 hours. The D-amidase activity of thethus obtained culture was 64 U/ml.

The cells obtained by centrifuging the culture were suspended in 1.9 lof 50 mM sodium phosphate buffer (pH 7.5). Then, the cells weredisrupted with a DYNO-MILL (laboratory mill model KDL, manufactured byW. A. Bachafen Maschinenfabrik). The cell suspension was centrifuged andthe resulting supernatant was subjected to DEAE-Sepharose Fast Flow(manufactured by Pharmacia Inc.) column chromatography which had beenequilibrated with 50 mM Tris-hydrochloride buffer (pH 7.5). Then,density gradient elution was carried out using the same buffer in whichthe rate of sodium chloride was increased from 0 to 0.4M. D-amidase waseluted in the fraction containing 0.2M sodium chloride The activefraction was further subjected to BUTYL TOYOPEARL (TSK-GEL 650° C.,manufactured by Toyo Soda Mfg. Co., Ltd.) column chromatography whichhad been equilibrated with 50 mM Tris-hydrochloride buffer (pH 7.5)containing 20% saturated ammonium sulfate. Then, density gradientelution was carried out using the same buffer in which the rate ofsaturated ammonium sulfate was decreased from 20% to 0.1%. D-amidase waseluted in the 15 to 10% saturated ammonium sulfate fractions. Afterdesalting the active fraction with UF membrane (SIP-1013, manufacturedby Asahi Chemical Co., Ltd.), glycerol was added to the active fractionin a concentration of 25% (v/v). The fraction was then subjected toDEAE-Trisacryl LS (manufactured by Reactifs IBF Soc. Chim) columnchromatography which had been equilibrated with 50 mM Tris-hydrochloridebuffer (pH 7.5) containing 25% (v/v) glycerol. Then, density gradientelution was carried out using the same buffer in which the rate ofsodium chloride was increased from 0 to 0.4M. D-amidase was eluted inthe fractions containing 0.2M sodium chloride.

The D-amidase activity of the thus obtained enzyme was 6.0×10⁴ U. Theenzyme showed a single band of molecular weight of about 50,000 inSDS-polyacrylamide gel electrophoresis.

The specific activity and Km value of the enzyme to D-α-alanineamide andL-α-alanineamide are shown in Table 1.

                  TABLE 1                                                         ______________________________________                                                  D-α-Alanineamide                                                                     L-α-Alanineamide                                 ______________________________________                                        Specific Activity                                                                         1800           17.4                                               (U/mg protein)                                                                Km (mM)     4.2            26.1                                               ______________________________________                                    

In FIG. 1, the activity with change in pH is shown in terms of relativeactivity, when defining the enzyme activity at pH 7.5 as 100%. Theenzyme had the optimum pH of 7 to 8 at 30° C.

In FIG. 2, the activity with change in temperature is shown in terms ofrelative activity, when defining the enzyme activity at 37° C. as 100%.As shown in FIG. 2, the enzyme had the optimum temperature of 40° to 45°C. at pH 7.5.

The pH stability of the enzyme was determined by the following method.

To 0.95 ml of various buffer solutions having pH of about 6 to 10containing 25% glycerol, 0.05 ml of 50 mM Tris-hydrochloride buffer (pH7.5) containing 19.2 U of the enzyme was added. The mixtures wereallowed to stand at 30° C. for 2 hours Then, 0.05 ml of each enzymesolution was added to 0.5 ml of D-α-alanineamide solution [50 mMphosphate buffer (pH 7.5) containing 12.5 g/l of D-α-alanineamide and25% (v/v) glycerol], incubated at 37° C. for 30 minutes, and terminatedby addition of 0.1 ml of 6N hydrochloric acid. The amount of D-α-alanineformed during the incubation was quantitatively determined. The enzymeactivity was expressed in terms of relative activity, when defining theenzyme activity before standing at 30° C. for 2 hours as 100%. Theresults are shown in FIG. 3. As shown in FIG. 3, the enzyme was stablein a pH range of 6.5 to 10.

The temperature stability of the enzyme was determined by the followingmethod.

50 mM Tris-hydrochloride buffer (pH 7.5), 0.5 ml, containing 192 U ofthe enzyme was diluted to 10-fold with 50 mM phosphate buffer (pH 7.5)containing 25% glycerol. The diluted solution, 0.5 ml, was taken,allowed to stand for 10 minutes at various temperature, and immediatelythereafter ice-cooled. To 0.5 ml of D-α-alanineamide solution [50 mMphosphate buffer (pH 7.5) containing 12.5 g/l of D-α-alanineamide and25% (v/v) glycerol], 0.05 ml of the enzyme solution was added, incubatedat 37° C. for 30 minutes, and terminated by addition of 0.1 ml of 6Nhydrochloric acid. The amount of D-α-alanine formed during theincubation was quantitatively determined. The enzyme activity wasexpressed in terms of relative activity, when defining the enzymeactivity before standing at various temperatures for 10 minutes as 100%.The results are shown in FIG. 4. As shown in FIG. 4, the enzyme wasinactivated when allowed to stand for 10 minutes at temperatures above60° C.

EXAMPLE 2

BYG medium (150 ml) having the same composition as in Example 1 waspoured into each of 2 l-flasks provided with baffles, and sterilized at120° C. for 20 minutes. Then, one loopful of Arthrobacter sp. H-4904growing on a bouillon slant was inoculated into each of the media andcultured at 30° C. for 20 hours with shaking. The resulting culture wasused as a seed culture.

Separately, a medium containing 3% of glucose, 2% of corn steep liquor,0.5% of peptone, 1% of NaCl, 2% of (NH₄)₂ SO₄, 0.3% of MgSO₄.7H₂ O,0.001% of FeSO₄.7H₂ O, 0.0001% of MnSO₄.7H2O and 0.6% ofDL-α-alanineamide and having a pH of 7.2 was prepared, and 1.5 l of thethus prepared medium was poured into a 3 l-jar fermenter and sterilizedat 120° C. for 20 minutes. Then, 150 ml of the seed culture wasaseptically introduced into the medium and cultured with agitation andaeration (800 rpm, 1 vvm) at 30° C. for 24 hours. The thus obtainedculture was centrifuged at 4° C. for 10 minutes at 5,000 rpm. A solutioncontaining 420 g of DL-α-alanineamide (592 g of hydrochloride thereof),15.6 g of NaH₂ PO₄.2H₂ O and 35.8 g of Na₂ HPO₄.12H₂ O in deionizedwater was added to 10 g of the thus obtained wet cells to make the totalvolume of 2 l. The pH of the resulting mixture was adjusted to 6.7 with10N NaOH. The hydrolysis reaction was carried out at 38° C. for 10 hourswith gentle stirring. The mixture was kept at pH 6.7 with 6N HCl duringthe reaction.

After completion of the hydrolysis reaction, the amount of D-α-alanineand the amount of L-α-alanineamide in the reaction mixture werequantitatively determined. The results are shown in Table 2.

                  TABLE 2                                                         ______________________________________                                                  Titer Reaction yield                                                                             Optical purity                                             (g/l) (%)          (%)                                              ______________________________________                                        D-α-alanine                                                                         105     99           99.3                                         L-α-alanineamide                                                                    105     --           99.0                                         ______________________________________                                    

Then 1 l of the reaction mixture was taken and the pH of the reactionmixture was adjusted to 4.5 and passed through a column packed with 3 lof Diaion SKlB (NH₄ ⁺ form) (product of Mitsubishi Kasei Corporation) toseparate D-α-alanine from L-α-alanineamide, and the respective fractionswere crystallized through condensation under reduced pressure forprecipitation, whereby 85 g of D-α-alanine (optical purity: 99.5% orhigher) and 75 g of L-α-alanineamide (optical purity: 99.5% or higher)were obtained.

EXAMPLE 3

The hydrolysis reaction was carried out in the same manner as in Example2 except that 210 g of D-α-alanineamide was used in place ofDL-α-alanineamide.

As a result, 103 g/l D-α-alanine (optical purity: 99.1%) was formed andaccumulated at the end of the reaction.

EXAMPLE 4

A desired mutant from Arthrobacter sp. H-4904 as the parent strain wasobtained as follows.

The parent strain was cultured in an NB medium [a medium containing 20 gof bouillon powder (product of Kyokuto Co.) and 5 g of yeast extract(product of Difco Co.) in 1 l of water, adjusted to pH 7.2 with NaOH] at30° C. for one day. The cells were recovered and suspended in a 0.1NTris-maleic acid buffer solution (pH 6.0) at a concentration of 10⁸cells/ml, and then 400 μg/ml N-methyl-N'-nitro-N-nitrosoguanidine (NTG)was added to the suspension. The suspension was incubated at roomtemperature for 30 minutes. The cells were thoroughly washed withphysiological saline, smeared onto an NB agar medium (NB mediumsupplemented with 20 g of agar) and cultured at 30° C. for 1 to 6 days.The developing colonies were smeared on another NB agar medium andcultured at 30° C. for 1 to 2 days

One loopful of the thus obtained strain was inoculated in a 250ml-Erlenmeyer flask containing 40 ml of a BYG medium sterilized at 120°C. for 20 minutes and culturing was carried out at 30° C. for 21 hourswith shaking. The thus obtained culture was centrifuged at 5,000 rpm for10 minutes and the thus recovered cells were suspended in a 50 mM sodiumphosphate buffer solution (pH 7.5) containing D-α-alanineamidehydrochloride to make a cell concentration of 2 g/l as wet cells. TheD-α-alanineamide hydrochloride concentration was adjusted to make thefinal concentration of D-α-alanineamide of 25 g/l. After the reaction at38° C. for one hour, the thus formed D-α-alanine was quantitativelydetermined through high performance liquid chromatography under the sameconditions as in Example 1 to calculate the D-amidase activity per gramof the wet cells.

The D-amidase activity was expressed by units per gram of wet cell.Arthrobacter sp. H-4904 was used as control and cultured in the samemanner as above except that 2 g/l DL-α-alanineamide was added to themedium, and the D-amidase activity was likewise determined.

In this manner, Arthrobacter sp. H-7095 was obtained as a mutant havingthe substantially same D-amidase activity as that of its parent strain,when cultured in a medium containing no inducing substance. The resultsare shown in Table 3.

                  TABLE 3                                                         ______________________________________                                                                  D-amidase activity                                  Strains    DL-α-Alanineamide                                                                      (U/g wet cells)                                     ______________________________________                                         Arthrobacter sp.                                                                        Added          666                                                 H-4904                                                                        Arthrobacter sp.                                                                         None           633                                                 H-7095                                                                        ______________________________________                                    

EXAMPLE 5

The hydrolytic reaction was carried out in the same manner as in Example2, except that the cells of Arthrobacter sp. H-7095 were used which hadbeen cultivated in a medium containing no DL-α-alanineamide.

The results are shown in Table 4.

                  TABLE 4                                                         ______________________________________                                                  Titer Reaction yield                                                                             Optical purity                                             (g/l) (%)          (%)                                              ______________________________________                                        D-α-alanine                                                                         104     99           99.2                                         L-α-alanineamide                                                                    105     --           99.0                                         ______________________________________                                    

EXAMPLE 6

The hydrolytic reaction was carried out in the same manner as in Example5, except that 210 g of D-α-alanineamide was used in place ofDL-α-alanineamide. As a result, 104 g/l D-α-alanine (optical purity:99.3%) was formed and accumulated at the end of reaction.

EXAMPLE 7

The culturing was carried out using Arthrobacter sp. H-7095 in thesimilar manner as in Example 5 to obtain 90 g of wet cells. The cellswere washed with physiological saline and suspended in distilled waterto make the total volume of 50 ml. A solution of 9 g of sodium arginate(Fuji Chemical Co., Ltd.) in 300 ml of distilled water was added to thesuspension. The resulting mixture was dropwise added to 2% calciumchloride solution to give immobilized cells having a diameter of about 2mm. Using the immobilized cells (25 g/l), the reaction was carried outin the same manner as in Example 2. As the result, 105 g/l D-α-alaninewas formed and accumulated (optical purity: 98.2%).

EXAMPLE 8

The culturing was carried out using Arthrobacter sp. H-7095 in thesimilar manner as in Example 5 to obtain 50 g of wet cells. The cellswere suspended in 10 mM phosphate buffer (pH 7.0) to make the totalvolume of 200 ml. The cells contained in the suspension were disruptedwith a homogenizer (Nissei Excel, Autohomogenizer) at 15,000 rpm for 20minutes under ice-cooling. The resulting suspension was centrifuged at4° C. for 20 minutes at 12,000 rpm to give the supernatant as the cellextract. The supernatant (100 ml) was brought into contact with 20 ml ofHPA-75 (product of Mitsubishi Kasei Corporation) which had beenequilibrated with 10 mM phosphate buffer (pH 7.0) at 5° C. for 24 hours.To the suspension of HPA-75 was added 0.5% glutaraldehyde solution, andthe mixture was incubated at 4° C. for 120 minutes to render HPA-75crosslinked with D-amidase. Then, the mixture was washed 3 times with 10mM phosphate buffer (pH 7.0) to give immobilized D-amidase. The specificactivity of the immobilized product was 550 U/ml. Using the immobilizedproduct (20 ml/l), the reaction was carried out in the similar manner asin Example 2. As a result, 104 g/l D-α-alanine was formed andaccumulated (optical purity: 99.2%).

What is claimed is:
 1. A process for producing at least one ofD-α-alanine and L-α-alanineamide, which comprises carrying out anenzymatic hydrolysis in an aqueous medium containing DL-α-alanineamidein the presence of a culture, cells, or a treated product thereof, of amicroorganism belonging to the genus Arthrobacter and capable ofproducing D-amidase having the following physicocochemical properties:a)activity and substrate specificity: it hydrolyzes D-α-alanineamide intoD-α-alanine; b) optimum temperature: 40° to 45° C. at pH of 7.5; c) heatstability: inactivated at a temperature above 60° C. for 10 minutes; d)pH stability: stable within a range of pH 6.5 to 10.0 at 30° C.; e)molecular weight: 50,000±5,000 daltons by SDS-polyacrylamideelectrophoresis; f) activation: no co-enzyme required for activation; g)isoelectric point: pH 5.2±0.3;and recovering at least one of D-α-alanineand L-α-alanineamide from the resulting mixture.
 2. The processaccording to claim 1, wherein the concentration of the cells is 1 to 50g/l as wet cells.
 3. The process of claim 1, wherein the concentrationof D-α-alanineamide is 1 to 300 g/l.
 4. The process of claim 1, whereinthe concentration of DL-α-alanineamide is 1 to 500 g/l.
 5. The processof claim 1, wherein the microorganism is Arthrobacter sp. H-4904 (FERMBP-1649).
 6. The process of claim 1, wherein the microorganism isArthrobacter sp. H-7095 (FERM BP-1773).
 7. A process for producingD-amidase having the following physiocochemical properties:a) activityand substrate specificity: it hydrolyzes D-α-alanineamide intoD-α-alanine; b) optimum temperature: 40° to 45° C. at Ph of 7.5; c) heatstability: inactivated at a temperature above 60° C. for 10 minutes;inactivated at a temperature above 60° C. for 10 minutes; d) pHstability: stable within a range of pH 6.5 to 10.0 at 30° C.; e)molecular weight: 50,000±5,000 daltons by SDS-polyacrylamideelectrophoresis; f) activation; no co-enzyme required for activation; g)isoelectric point; pH 5.2±0.3;said process comprises culturing amicroorganism capable of producing D-amidase and belong to the genusArthrobacter in an aqueous nutrient medium containing assimilablesources of carbon, nitrogen, and inorganic substances; and recoveringsaid D-amidase.
 8. The process according to claim 7, wherein the mediumcontains DL-α-alanineamide, D-α-alanineamide or L-α-alanineamide as aninducing substance.
 9. The process according to claim 7, whereinD-amidase is recovered from cells in the culture.
 10. The process ofclaims 7, 8 or 9, wherein the microorganism is Arthrobacter sp. H-4904(FERM BP-1649).
 11. The process according to any one of claims 7, 8 or9, wherein the microorganism is Arthrobacter sp. H-7095 (FERM BP-1773).12. A biologically pure culture of a microorganism of the genusArthrobacter having all the identifying characteristics of Arthrobactersp. H-4904 (FERM BP-1649).
 13. A biologically pure culture of themicroorganism of the genus Arthrobacter having all the identifyingcharacteristics of Arthrobacter sp. H-7095 (FERM BP-1773).